This invention relates to bacteriophages useful for the treatment of bacterial infections, especially mucosal bacterial infections such as Helicobacter pylori infections.
Bacteriophages and Antibiotic Resistance
Resistance to antibiotics is a global problem of increasing medical and economical importance. There is thus a great need for alternative methods to eradicate bacteria which will circumvent the problem of such resistance.
A bacteriophage, or phage, is a virus which specifically infects bacteria. Phages bind to their host bacterium and transfer genes encoding various phage proteins. They utilize the protein-synthesizing machinery, amino acids etc., and the energy provided by the host bacterium for their replication (Maloy et al. (eds.): Microbial genetics. Jones and Bartlett Publishers, 1994).
Most phages lyse or by other mechanisms destroy specific strains of bacteria. The present invention stems from the realisation that genetic modification of phages, in particular filamentous bacteriophages, offers a means for designing new bacterium-specific phages capable of eradicating certain bacteria, e.g. Helicobacter pylori, and having the potential to overcome problems related to antibiotic resistance.
Filamentous Phages
E. coli cells bearing hair-like F-pili are hosts for filamentous phages such as M13, fd and f1. These Ff (F pili, filamentous) phages are nearly identical in sequence and behaviour (Rashed and Oberer (1986) Microbiological reviews 50, 401-427; Kornberg and Baker, in: DNA Replication, p. 557-570, W.H. Freeman and Co., New York 1992). Ff phages alone among the bacterial viruses do not produce a lytic infection, but rather induce a state in which the infected host cells produce and secrete phage particles without undergoing lysis.
The single-stranded genome of phage M13 encodes 10 different proteins. The DNA is enclosed in a protein coat comprised of approximately 2700 copies of the gene 8 protein (g8p). A viable M13 phage also expresses five copies of the 43 kDa gene 3 protein (g3p) on its tip, which protein is responsible for adsorption to E. coli pili. The gene 3 protein is anchored to the virus coat via the C-terminal part of the polypeptide chain, whereas the N-terminal globular domain is exposed and mediates the attachment of the phage to the tip of a host F pilus. By electron microscopy, the adsorption complex appears as a xe2x80x9cknob-on-stemxe2x80x9d structure at one end of the phage. During infection, the leader sequences of g3p and g8p direct the transport of these proteins into the inner membrane of the bacterial cell.
The Ff phages have gained popularity as cloning vectors because they have no physical constraints limiting the length of DNA that can be packaged and because they allow the easy purification of single-stranded DNA. A phagemid is a vector which carries both the M13 (single-stranded) and plasmid (double-stranded) origins of replication. Phagemids can be grown as plasmids or packaged as recombinant M13 phage with the aid of a helper phage such as M13K07 (Veira and Messing (1987) Methods in Enzymol. 153, 3-11).
Recombinant Antibody Production
Antibody molecules contain discrete fragments which can be isolated by protease digestion or produced by recombinant techniques. One such fragment is the Fv (fragment variable) which is composed only of the VL and VH regions of the antibody. In U.S. Pat. No. 4,946,778 a recombinant version of the Fv fragment, designated single-chain Fv (ScFv), is described, where the two variable regions are artificially joined with a neutral linker and expressed as a single polypeptide chain.
A technology for recombinant antibody production has been developed by McCafferty and coworkers (McCafferty (1990) Nature 348, 552-554; Winter and Milstein (1991) Nature 349, 293). This approach relies on a phage-display system in which VH (variable heavy) and VL (variable light) genes are cloned into a phage vector whereafter fragments of antibodies are expressed as fusion proteins displayed on the phage surface. With this approach, antibodies of defined specificity and affinity can be selected from a population. It has been suggested that antibodies isolated and manufactured in prokaryotic systems should be called xe2x80x9ccoliclonalxe2x80x9d antibodies (Chiswell and McCafferty (1992) Trends in Biotechnology 10, 80-84).
The commercially available phagemid pCANTAB5 is designed such that antibody variable region genes can be cloned between the leader sequence and the main body of the M13 gene 3. The g3p leader sequence directs transport of the resulting fusion protein to the inner membrane and/or periplasm of E. coli where the main g3p domain attaches the fusion protein to the tip of the assembling phage. The expression of the antibody-g3p gene is controlled by an inducible lac promoter on the phagemid.
Helicobacter pylori Infection
It is widely accepted that the bacterium Helicobacter pylori is the main cause of gastric and duodenal ulcer, responsible for 84% and 95%, respectively, of reported cases (Kuipers, E. L. et al. (1995) Aliment. Pharmacol. Ther. 9 (suppl.2), 59-69). H. pylori colonises the wall of the stomach, protected from the acid environment by a layer of mucus which lines the stomach wall, and by a metabolic process which enables the organism to secrete ammonia to neutralise acid.
Conventional antibiotic treatment appears to have little effect on H. pylori. This is probably due to: (i) poor access of the antimicrobial agent to the organism which is not directly exposed to the blood circulation; and (ii) rapid passage of many oral antibiotics through the stomach, or degradation of such antibiotics in the acid conditions of the stomach.
The purpose of the present invention is to provide new forms of treatment for eradication of bacteria, especially eradication of bacteria responsible for mucosal bacterial infections such as Helicobacter pylori. In particular, it provides filamentous bacteriophages genetically modified to have binding specificity towards another bacterial host for use in therapy.
Methods of treatment of mucosal bacterial infections based on recombinant phages are believed to be superior to conventional antibiotic treatment for several reasons, e.g. the following:
it will be possible to eradicate bacteria resistant to conventional antibiotics;
the high specificity of the recombinant phage towards specific bacterial species;
propagation of the phage is self-limiting;
in the case of Helicobacter pylori infections, the motility of Helicobacter pylori could help to distribute the phage to all parts of the gastric mucosa.
In the present description and examples, the terms xe2x80x9cstandard protocolsxe2x80x9d and xe2x80x9cstandard proceduresxe2x80x9d are to be understood as protocols and procedures found in an ordinary laboratory manual such as: Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
In a first aspect, this invention provides a modified bacteriophage for use in the treatment or prophylaxis of a bacterial infection, which bacteriophage presents at its surface a recombinant protein comprising
(i) a first component derived from a bacteriophage surface protein; and
(ii) a second component comprising variable region sequences of an antibody to provide a bacterial antigen binding site, said second component rendering said bacteriophage capable of binding to and thereby inhibiting growth of bacterial cells involved in the etiology of said infection.
The said modified bacteriophage can e.g. be a modified filamentous phage, such as a modified M13 phage.
The said bacterial infection can e.g. be a mucosal bacterial infection such as Helicobacter pylori infection. However, the present invention is not restricted to phages capable of incapacitating Helicobacter pylori cells, but rather comprises phages with altered properties which can be used for incapacitating a wide range of bacteria. It will be understood that a phage according to the invention which is specific for any bacterial species can be prepared by the skilled person on the basis of the present disclosure. Phages according to the invention are suitable for treatment of any mucosal bacterial infection accessible to the outside world. Examples of such mucosal epitheliums are nasal, lung, gastrointestinal tract, urinary bladder and vagina.
Examples of other bacterial infections which could be treated with phages according to the invention are:
infections in the urinary tract by E. coli, Staphylococcus saprophyticus, Klebsiella spp, Proteus spp or Pseudomonas aeruginosa; 
vaginal infections by Clamydia;
nose/toncillar/lung infections by Streptoccus, Staphylococcus, Haemophilus influence, Pneumococcus or Mycoplasma pneumonic; 
infections in the gastrointestinal tract by Salmonella, Shigella, Yersinia, Campylobacter jejuni, Campylobacter coli, Helicobacter, Vibrio cholera or E. coli. 
The said first component of the recombinant protein mentioned above can preferably be derived from the protein responsible for adsorption of the unmodified form of said bacteriophage to bacterial pili, e.g. the g3p protein from a M13 phage.
The said second component of said recombinant-protein can e.g. comprise a recombinant single-chain Fv (ScFv) polypeptide. Consequently, the said recombinant protein can e.g. be a g3p-ScFv fusion protein.
In a preferred form, the bacteriophage according to the invention is a bacteriophage for use in the treatment or prophylaxis of Helicobacter pylori infection wherein the antibody variable region sequences of said recombinant polypeptide are variable region sequences of a monoclonal antibody selected from the monoclonal antibodies of hybridoma cell lines 5F8 (ECACC No.95121524), 2H6 (ECACC No.95121526) and 5D8 (ECACC No.95121527).
Thus, the bacteriophage according to the invention can e.g. be the modified M13 bacteriophage designated B8 deposited at the NCIMB under accession number NCIMB 40779, or a derivative thereof which retains the ability to bind and infect Helicobacter pylori. 
Phages with the desired properties can be obtained by e.g. one of the following methods:
(a) Screening naturally occurring phages, or phage libraries containing phages expressing a multitude of variable antibody fragments. Phage libraries may be obtained e.g. from immune cells, from a large number of individuals. Due to the vast genetic variability, such large phage libraries are likely to comprise the desired, specific phage directed towards bacteria, to which the individuals previously have been exposed.
(b) Development of mutations in existing phages. Mutations occur in all living organisms including phages. The frequency of mutations may be increased, e.g. by chemical means or by means of short wavelength electromagnetic irradiation.
(c) Directed genetic modification of one or more amino acids, or other modifications of e.g. carbohydrate or lipid components, of the binding region of the phage, in order to increase the desired properties of the natural or recombinant phage. An example of this approach is further described in the experimental section. A bacteriophage according to the invention can thus be produced by a method comprising (a) isolating an antibody against a bacterial cell; (b) isolating the DNA encoding for a variable region of the heavy and light chains of the said antibody; and (c) introducing the said DNA into phage DNA so that the said antibody regions will be expressed on the surface of the phage.
In another aspect, the invention provides a pharmaceutical composition comprising a bacteriophage according to the invention in admixture with a pharmaceutically acceptable carrier or excipient.
Examples of suitable means of administration of phages according to the invention include:
spray for nasal and lung applications;
pre-treatment with omeprazole followed by phages suspended in bicarbonate buffers for the treatment of gastrointestinal mucosae;
mixtures of muco-adhesive gels (i.e. cellulose-based gels, polycarbophil, poloxamer etc.) for the gastric mucosa and vaginal mucosa;
bicarbonate buffers for use in the urinary bladder.
The number of phages to be administered can be determined by the skilled person. Depending on the type of infection, the number of phages to be administered can range from 104 to 1010.
In yet another aspect, the invention provides a method for treatment of a bacterial infection in a mammal which comprises administering a bacteriophage or pharmaceutical composition according to the invention. The said bacterial infections can e.g. be mucosal bacterial infections such as Helicobacter pylori infections. Included in the invention is also the use of a bacteriophage according to the invention in the manufacture of a medicament for the treatment or prophylaxis of a mucosal bacterial infection, e.g. Helicobacter pylori infections. Further aspects of the invention are a hybridoma selected from 5F8 (ECACC No.95121524), 2H6 (ECACC No.95121526) and 5D8 (ECACC No.95121527), as well as a monoclonal antibody selected from the monoclonal antibodies produced by the said hybridomas. Hybridoma technology, in which antibody-producing B-cells from immunized animals are fused with myeloma cells, and resulting hybridoma cell lines producing the desired antibody are selected, is well known in the art.